Monitoring genetic fidelity vs somaclonal variation in Norway spruce (Picea abies) somatic embryogenesis by RAPD analysis

Summary Somaclonal variation, which is a welcome source of genetic variation for crop breeding, is unwanted when direct regenerants have to be used in tissue culture mass propagation (eg. in many forest trees), or in the regeneration of genetically transformed plants. Random amplified polymorphic DNA (RAPD) was used to analyse somatic embryos and plants regenerated from embryogenic cell lines in Norway spruce, Picea abies (L.) Karst. RAPD facilitated the identification of clones, as material from the same cell lines shared identical patterns of amplified fragments, whereas regenerants from different cell lines were easily distinguishable by their respective patterns. For comparisons with explant donor genotypes, cell lines were initiated from cotyledons. Some of the seedlings that had parts of their cotyledons removed were grown on as control plants. Somatic embryos regenerated from cotyledon cell lines showed no aberrations in RAPD banding patterns with respect to donor plants. We conclude that gross somaclonal variation is absent in our plant regeneration system.Abbreviations ESM embryogenic suspensor mass - RAPD random amplified polymorphic DNA - RFLP restriction fragment length polymorphism - (2,4-dichlorophenoxy)acetic acid 2,4-D - 1-naphthaleneacetic acid NAA     

Linamarin content and genetic stability of cassava plants derived by somatic embryogenesis

A protocol was established for high frequency cyclic somatic embryogenesis for different varieties of cassava. An efficient plant regeneration system was developed for the high cyanogenic variety PRC60a. Linamarin content and linamarase activity were determined in various tissues of secondary somatic embryos and regenerated plants of PRC 60a. Both linamarin and linamarase activity were not detected in embryogenic callus, roots induced from callus and somatic embryo tissues. The stems and leaves of regenerated plants (in vitro) and storage roots and leaves of mature plants (in vivo), however, contained variable amounts of linamarin and linamarase activity whereas in the non storage root tissues (in vitro) only linamarin was detected. The present study suggested that the linamarin biosynthetic pathway may be absent or not switched on in the embryogenic callus and somatic embryos. The ploidy level and somatic chromosome number of the regenerated plants were found to be same as the source plants. The availability of this regeneration system would be useful not only for investigating cyanogenesis but also for genetic manipulation in cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Plant regeneration in sweet potato (Ipomoea batatas L., Convolvulaceae)

The application of new techniques for improvement of sweet potato crops, particularly including the exploitation of somaclonal variation, gene transfer by genetic transformation and somatic hybridization, requires the control of plant regeneration from tissue cultures. Shoots can easily be regenerated from explants of stems, petioles, leaves and roots, while callus cultures do not produce any shoots. The potential of somatic embryogenesis and plant regeneration via embryogenesis was evaluated for 10 cultivars of sweet potato. Protocols for plant regeneration from cultured protoplasts have also been developed. Since mesophyll was resistant to enzyme digestion, fragments of stems and petioles, callus and cell suspensions were used as source of protoplasts of sweet potato. Series of transfers of protoplast-derived calluses, particularly those which had been obtained from in vitro plants, to media containing a high level of zeatin resulted in successful formation of shoots in only two sweet potato cultivars. In addition, the embryogenic potential was irreversibly lost through protoplast culture, since protoplasts isolated from embryogenic cell suspensions developed into non-embryogenic callus. Consequently, an alternative protocol is being successfully developed to improve plant regeneration from cultured protoplasts of sweet potato, involving first root formation from which shoots can then be regenerated. Preliminary evaluation in field conditions in Gabon revealed that plants regenerated from cultured protoplasts exhibited a great genetic variability in their growth and tuber formation in particular. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic control of somatic embryogenesis in alfalfa (Medicago sativa L. cv. Adriana)

Summary In alfalfa (Medicago sativa) regeneration is genotype-specific. In order to study the genetic control of somatic embryogenesis and to constitute a synthetic cultivar characterized by its high regeneration ability, 2 embryogenic plants selected from the cv. Adriana were selfed, intercrossed and also crossed in both directions with 5 non-embryogenic genotypes of the same cultivar.Progenies of all crosses were scored for their regeneration ability and results indicate that somatic embryogenesis is under the control of 2 dominant loci. However some non-embryogenic genotypes prevent regeneration when crossed with embryogenic ones and this characteristic is not under the control of a single dominant gene.When plants chosen for their capacity to regenerate within F₁ and S₁ progenies were freely intercrossed the regeneration efficiency dropped to 2% (1 plant out of 50). This result indicates that if the genetic background of the population is changed the regeneration is greatly affected and therefore some other mechanism could play a role in determining plant regeneration.     

RAPD variability in rice (Oryza sativa L.) plants derived from desiccation-tolerant calli

Genetic modification from selfed progenies of 18 rice (Oryza sativa L.) plants regenerated from callus tissues which survived desiccation, were investigated at the DNA level using the random amplified polymorphic DNA (RAPD) method. Twelve 10-mer random primers were used to amplify DNA of progenies from the regenerated plants, and a total of 228 PCR products and 1780 DNA fragments were obtained by primers, generating between four to thirteen major bands. The size of the amplified fragments ranged from 0.2 to 2.55 kb. The results showed that 10 out of 12 primers produced polymorphic bands, two primers (RA31 and RA185) showed no polymorphism among plants tested. A dendrogram of the genetic distance was constructed based on their polymorphism, demonstrating that somaclonal variation exists in rice plants regenerated from callus which survived the desiccation treatment. Part of this variation can be useful in rice breeding. This revised version was published online in August 2006 with corrections to the Cover Date.     

Somatic embryogenesis and plant regeneration of Tripsacum dactyloides L.

Summary This article reports the culture and plant regeneration of Tripsacum dactyloides. Mature embryos of Tripsacum dactyloides dactyloides were used to obtain embryogenic callus cultures. Currently, 180 normal plants have been regenerated from these cultures. Callus was initiated on MS medium supplemented with dicamba (10 mol or 20 mol) and sucrose (3% or 6%), and plants were regenerated on hormone free MS medium containing 2% sucrose. No significant differences were found in callus initiation frequency or in embryogenic response of cultures on the four combinations of sucrose and dicamba tested. The embryogenic cultures have been maintained for 9 months (12 subcultures) and have retained regeneration capacity. Plants regenerated from tissue culture of maize-by-Tripsacum hybrids could be useful in maize improvement.     

甘薯胚性细胞悬浮培养系的建立

地甘薯胚性细胞悬浮增减系的进行了研究。将１２个基因的长约０．５ｍｍ的茎尖培养在含有０．２ｍｇ／Ｌ或２．０ｍｇ／Ｌ２，４－Ｄ的ＭＳ培养基上，形成了胚性愈伤组织。胚性愈伤组织的形成率因基因型和２，４－Ｄ深度不同而很大差异，为０－７５．７％。一方面，将胚性愈伤组织继续增减在含有２，４－Ｄ的ＭＳ培养基上，它们形成了处于各发育时期的体细胞胚。将具有体细胞胚的胚性愈伤组织转移到ＭＳ基本培养基上，体细胞胚发育成     

低酚陆地棉直接体细胞胚胎发生和植株再生

选用低酚陆地棉无菌苗下胚轴为材料进行全固体组织培养,直接诱导获得了胚性愈伤组织,并进一步分化为再生植株.结果表明,激素是影响棉花直接体细胞胚胎发生的重要因素.MSB培养基中添加2,4-D有利于愈伤组织的形成,却不能直接诱导获得胚性愈伤组织.MSB培养基中添加IBA和BA也不能直接诱导获得胚性愈伤组织.MSB培养基附加适当浓度的IBA和KT能直接诱导出胚性愈伤组织.最适激素组合(1.0 mg/L IBA,0.5 mg/L KT)能使诱导棉花下胚轴产生大量胚性愈伤组织,并且在3个月内就可肉眼观察到不同发育时期的胚.MSB培养基中附加1.0 g/L谷氨酰胺和0.5 g/L天门冬酰胺有利于胚萌发成苗.本研究建立了简便高效的棉花直接体细胞胚胎发生和植株再生培养体系,从胚性愈伤组织诱导到植株再生约需5～6个月时间.     

The role of nickel on somatic embryogenesis in Setaria italica L. in vitro

Nickel (0.13, 0.25, 0.5, 1.0 and 1.5 mg/l) increased the efficiency of somatic embryogenesis in leaf base and mesocotyl derived calli of Setaria italica. A lower concentration of nickel in the culture media promoted long-term maintenance of embryogenic calli that regenerated into plantlets. The plants obtained from embryogenic calli grown on Ni-containing medium showed tolerance to nickel. The growth of embryogenic callus was the maximum at 1.5 mg/l as compared to a lower or a higher concentration of Ni. Use of nickel may help in the induction of high frequency somatic embryogenesis in Setaria italica. This revised version was published online in July 2006 with corrections to the Cover Date.     

Progress made in FEC transformation of cassava

In cassava friable embryogenic callus (FEC) has been used to obtain transgenic plants using particle bombardment, electroporation, and Agrobacterium tumefaciens. FEC cultures have been obtained in 6 of the10 tested genotypes. In all genotypes FEC could be regenerated into plants,however the efficiency differed between the genotypes. Almost all plants regenerated from 6 months old FEC cultures of TMS604444, Adira 4,Thai 5 and M7 were morphological similar to control plants. However, in R60 and R90 a large number of plants were not identical to control plants. Older FEC lines of TMS60444 have a reduced ability to regenerate plants and the plants show somaclonal variation. Somaclonal variation is observed in the same extend in transgenic and non-transgenic plants. The origin of this variation is both genetic and epigenetic. Luciferase based selection is less efficient in producing transgenic lines than chemical selection. Furthermore Agrobacterium tumefaciens mediated transformation is much more efficient than particle bombardment with respect to the production of transgenic lines. A tentative model is introduced which best describes the effect of different selection regimes on the time period required to produce transgenic plants. Kanamycin and stringent luciferase selection required a shorter period of time than selection based on hygromycin, phosphinothricin or non-stringent luciferase. However, a more significant reduction of time was obtained if young instead of old FEC lines of genotype TMS60444 were used for genetic modification. In accordance to the model these young FEC lines of TMS60444 produced transgenic plants within 4 months with both Agrobacterium tumefaciens combined with kanamycin selection and particle bombardment combined with stringent luciferase selection. This revised version was published online in July 2006 with corrections to the Cover Date.     

High frequency plant-regeneration through direct shoot development and somatic embryogenesis from immature inflorescence cultures of finger millet (Eleusine coracana Gaertn)

Summary Plant regeneration from cultured immature inflorescence segments of Eleusine coracana was obtained by direct shoot development and somatic embryogenesis. Direct development of shoots from cultured inflorescence segments occurred on MS medium supplemented with 2,4-D in combination with zeatin. Inflorescences with well developed spikelets differentiated at a low frequency (<5%) from callus cultures initiated on media supplemented with 2,4-D in combination with zeatin or coconut water or picloram + kinetin. Somatic embryogenesis was also induced in callus cultures growing on MS + picloram + kinetin at the end of four passages. Supplementation of the media with different concentrations of sucrose showed 3% sucrose as the best concentration for plant differentiation from somatic embryos. The majority of the regenerated plants showed the diploid chromosome constitution in their root tips. The regenerants were in general shorter with an increased number of tillers compared to the control.Abbreviations CW Coconut water - 2,4-D 2,4-dichloro phenoxyacetic acid - Kn Kinetin - Z Zeatin     

Somatic Embryogenesis in Maize and Comparison of Genetic Variability Induced by Gamma Radiation and Tissue Culture Techniques

Sixteen inbred lines and one hybrid of manse were tested for their capability of somatic embryogenesis, and fully developed plants could be regenerated, from ten inbred, lines. The highest frequency of plant regeneration was expressed in the inbred line CHI 31, and when this line was crossed with a recalcitrant, non-regenerating line, the F₁ and BC hybrids were regenerable. The results of reciprocal crosses demonstrated that dominant nuclear genes and cytoplasmic factors are primarily responsible for the heritable determination of embryogenic callus proliferation and in vitro regeneration of maize plants. Somaclonal and radiation-induced variability was studied in maize to assess their nature and potential contribution to plant breeding., The inbred line CHI 31 possessing a high in vitro capacity of somatic embryo formation was used as experiments.] material. CHI 31 plants were selfed and twelve-day old zygotic embryos irradiated with ⁶⁰Co gamma radiation in situ. Mature caryopses were harvested and assigned as M₁ material. In another series, immature zygotic embryos (size 1.2—1.5 mm) were cultured in vitro on N-6 medium supplemented with 2,4-D (2.5 μM), and somatic embryos regenerated into plants; these were transplanted into soil and self-pollinated. Regenerants from non-irradiated cultures were grown as R₁ generation, while regenerants from irradiated explants were considered as M₁R₁ generation. The genetic variability was evaluated in the M₂, R₂ and M₂R₂ generations, respectively, and compared with a non-treated seed control. Irradiation induced a variety of chlorophyll and morphological variants segregating in the M; generation; however, the frequency of deviant types obtained in the R: generation (somaclonal variation) was significantly exceeding the one derived from the M₂ populations. The combination of expert irradiation and in vitro regeneration was most effective for the manifestation of chlorophyll and morphological o if types in the M₂R₂ generation, and increased drastically the frequency of early flowering variants. Differences in the segregation patterns of mutant phenotypes amonsister somaclones in the R₃ and M₃R₃ generations indicate a different genetic basis, of plants originating from the same explant. This phenomenon suggests a mutational sectoring of the callus during culture. Radiation induced and somaclonal variation exerted a similar spectrum of chlorophyll and morphological deviants.     

Trueness-To-Type and Yield Components of the Banana Hybrid Cultivar FHIA-18 Plants Regenerated Via Somatic Embryogenesis in a Bioreactor

Summary A population of 1,500 plants of the banana hybrid ‘FHIA-18’ (AAAB), regenerated from somatic embryos, which were multiplied in bioreactors, showed similar characteristics to plants propagated from shoot tip cultures both in the acclimatization stage and in field experiments carried out in Cuba. The plants originating from somatic embryos were similar to the plants obtained from shoot tips with respect to plant height, diameter of the pseudostem and number of suckers. Both groups of plants obtained from in vitro cultures were significantly different to the plants obtained from suckers during the flowering period of the mother plants, which was shortened by two months. The greater plant height and diameter of the pseudostem in the plants coming from somatic embryos and shoot tip is due to the effect of in vitro culture, and this was observed in different banana and plantain cultivars. During the second cycle of evaluation, the plants coming from the three propagation methods studied in this work had similar growth habits without significant differences in the majority of the morphological parameters evaluated. These results confirm that the difference obtained during the first cycle between the distinct populations is attributed to temporary changes. The original characteristics of the cultivar were evident from the second cycle of culture. Only 0.13% somaclonal variant was observed in the plants coming from somatic embryogenesis. These percentages are low taking into consideration that other propagated methods accept up to 5% variants in field conditions.     

A Regeneration System for Cotton Variety Xinluzao 45 via Somatic Embryogenesis

[Objective] The purpose of this study was to expand upland cotton genotypes suitable for regeneration via somatic embryogenesis, so as to provide excellent receptor materials for the genetic transformation of cotton. [Method] Six different hormone combinations were studied to establish a somatic embryo regeneration system for hypocotyl explants of Xinluzao 45, a widely planted variety in Xinjiang Province. [Result] Faint yellow callus was induced on a medium consisting of hormone combinations C1 (0.1 mg·L^－1 acetic acid dichlorobenzene oxide [2, 4-D] + 0.1 mg·L^－1 kinetin [KT]) or C5 (0.1 mg·L^－1 2, 4-D + 0.5 mg·L^－1 indo-3-butyric acid + 0.1 mg·L^－1 KT). The generated callus was able to successfully induce embryogenic callus on hormone-free medium containing doubled KNO3. The embryogenic callus then developed into somatic embryos and regenerated plants. Maximum rates of callus induction and regeneration were 92.2% and 40.3%, respectively. Although no significant difference was observed in the rate of callus induction of Xinluzao 45 hypocotyls between hormone combinations C1 and C5, the incidence of induced embryonic callus was higher on C1 than on C5. [Conclusion] The somatic embryo regeneration system developed in this study provides technical support that will facilitate Agrobacterium-mediated transformation of upland cotton Xinluzao 45.     

Somatic embryogenesis from floral tissue of cassava (Manihot esculenta Crantz)

The aim of this study was to examine the embryogenic potential of floral material of the cassava cultivar MCOL 1505. Macerated immature inflorescences were found to be highly embryogenic, with almost 78% of the explants producing somatic embryos. Somatic embryos were also produced from whole male florets and half florets although at much lower rates. No regeneration was obtained from anther, microspore or floret wall tissue. Somatic embryos derived from immature inflorescences were regenerated via organogenesis and the plants derived from this process were assessed in terms of phenotype and ploidy level. If haploid plants could be produced by this method, this would have significant implications in assisting traditional cassava breeding, as this would allow homozygosity to be reached more rapidly. In a crop such as cassava, which is highly heterozygous in nature, the use of haploids in a breeding programme could considerably shorten the time taken to produce new desirable cultivars. This is the first report on plant regeneration through somatic embryogenesis from floral tissue of cassava. This revised version was published online in July 2006 with corrections to the Cover Date.     

Efficient production of transgenic plants by Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz)

An efficient and reproducible method was developed for Agrobacterium-mediated transformation of embryogenic suspension cultures of cassava. LBA4404(pTOK233), containing the nptII, hph and gus marker genes, was used in the experiments. Chemical selection by means of kanamycin was used to establish 1037antibiotic resistant callus lines, of which 526 showed GUS expression. Of the 241 callus lines that were transferred to maturation medium 219formed somatic embryos. Thirty-seven of the 38 lines that were transferred to germination medium produced plants. GUS-positive plants could be obtained from 31 lines; in 14 of those lines 100% of the produced plants were GUS-positive, the remaining 17 lines yielded GUS-positive plants at an average of 72%. The transgenic nature of these plants was confirmed by Southern blot analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD markers linked to microspore embryogenic ability in Brassica crops

In order to identify the markers linked to microspore embryogenic ability in Brassica crops, RAPD segregation analyses were performed in a microspore-derived (MD) population and a F₂ population derived from F₁between ‘Ho Mei’ (high responsive parent in microspore embryogenesis) and ‘269’ (low responsive parent) in Chinese cabbage, and between ‘Lisandra’ (high responsive parent) and ‘Kamikita’ (low responsive parent) in oil seed rape. After 230 and 143 primers were screened, a total of 148 and 52markers were detected to be polymorphic between the parents in Chinese cabbage and oilseed rape, respectively. Twenty-seven percent of the markers in the MD population showed a significant segregation distortion in both crops. Of the markers showing segregation distortion in the MD population, 71–75% of the markers followed the expected Mendelian segregation ratio in the F₂ population. When the relationships between such distorted markers and microspore embryogenesis of the F₂ population were examined, 7 and 3 markers were identified to be associated with embryogenic ability in Chinese cabbage and oilseed rape, respectively. These markers showed additive effects on embryo yields, and the plants having more alleles of the high responsive parent produced higher embryo yields. These markers maybe useful in marker-assisted selection for improving microspore responsiveness straits in Brassica crops. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic variability measures among Bromus catharticus Vahl. populations and cultivars with RAPD and AFLP markers

The objectives of this study were to optimize RAPD and AFLP techniques in B. catharticus, and to determine the genetic variability of populations and commercial prairie grass cultivars with the aforementioned molecular markers. Two populations with contrasting morphological characteristics were evaluated from individual and bulked DNA samples using RAPD markers. Both analyses showed a similar information about inter population variability. Each accession was sampled by a single leaf bulk of 10 plants. Accession similarities were established with 276 RAPD and 714 AFLP bands using Jaccard similarity coefficient. The dendrogram of the accessions using RAPD markers showed that they shared high similarity values (>94%). A similar result was obtained with AFLP markers (similarity values >98%), revealing a narrow genetic basis in the analyzed accessions. Consequently, molecular characterization of germplasm should be considered in addition to morphological criteria, to choose the parental genotypes for breeding programs of this forage crop. The AFLP technique was more efficient to detect DNA polymorphism in our experiments and unique fingerprints were detected for all the accessions. RAPD is a simple and non expensive technique, suitable to estimate genetic similarity. This revised version was published online in August 2006 with corrections to the Cover Date.